An important mechanism for controlling GPCR signaling involves stimulus-dependent phosphorylation of the receptor, a process primarily mediated by G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of arrestins, which function in receptor/G protein uncoupling and subsequent GPCR endocytosis. In this application, the applicant proposes to continue his broad-based studies aimed at defining the role of GRKs in regulating cellular signaling. Three specific objectives are outlined in the application. 1. Elucidate the role of GRKs in GPCR regulation. The first aim focuses on characterizing the role of GRKs in regulating GPCR function, with emphasis on defining the specificity of GRK/GPCR interactions. Although GRKs have been broadly implicated in regulating receptor/G protein uncoupling, most studies have used in vitro and overexpression strategies to implicate GRKs in this process. Dr. Benovic proposes to use several approaches, including analysis of GRK translocation and the use of dominant negative mutant GRKs and antisense cDNA constructs, to elucidate the role and specificity of endogenous GRKs in intact cell systems. 2. Characterize the functional consequences of GRK association with GRK-interacting proteins. Recent studies have suggested the GRKs can interact with a number of cellular proteins in addition to GPCRs. The interacting proteins identified by the Benovic lab can be divided into four groups: 1) G protein subunits; 2) cytoskeletal proteins; 3) calcium binding proteins; and 4) others such as caveolin and PP2A. They hypothesize that these interactions play an important role in regulating GRK function and may be involved in mediating GPCR/GRK control of a diverse array of cellular effects. These interactions will be characterized using numerous strategies including detailed binding and functional analysis in vitro and co-immunoprecipitation and co-localization studies in intact cells. 3. Elucidate the role of GPCRs in regulating GRK interaction with other proteins. The third aim will attempt to integrate the first two aims and ascertain whether GRK interaction with GPCRs regulates kinase interaction with additional cellular proteins. Given the observation that GRK binding to GPCRs results in kinase activation, the applicant hypothesizes that such an activated GRK may interact with and/or phosphorylate a novel set of proteins. They will attempt to identify such proteins both in vitro and in intact cells. Overall, these studies should more clearly define the role of GRKs in regulating cellular signaling.